PROJECT SUMMARY Antibody affinity maturation can improve the affinity of antibodies for antigens by orders of magnitude, and is critical to immunity. In affinity maturation, somatic hypermutation (SHM) creates a library of mutant antibodies, and mutants with good binding affinity are selected to continue. It had previously been thought that SHM created near-random libraries for selection. However, in preliminary data presented here, we show that limited types of amino acid SHMs are explored for each antibody gene, and each gene has specific patterns of SHMs within the variable domain. For each germline V gene, some SHMs are generated with high frequencies (dominant SHMs) by the SHM machinery and are fixed in many mature antibody lineages with diverse antigen specificities, suggesting that such SHMs play specific roles beneficial to antibody structure and function. In this project, we propose to use approaches of structural biology, biophysics, and bioinformatics to understand how dominant SHMs affect antibody affinity maturation. In Aim 1, we will use x-ray crystallography and experimental biophysical measurements to study how dominant SHMs, observed at positions of human IGHV1-2 and IGHV1-69 genes, alter the stability, solubility, conformation, and binding affinity of unmutated- precursor and mature antibodies. In Aim 2, we will perform molecular dynamics simulation and develop methods to analyze simulation trajectories to investigate the effects of dominant SHMs on antibody conformational dynamics and flexibility. We will also use datasets from public databases and results from Aim 1 to evaluate the accuracies of popular bioinformatics methods to predict the effects of SHMs on antibody stability and solubility, and will search for protocols to maximize accuracies of predictions. The optimal methods and protocols identified will be used to develop a bioinformatics pipeline to understand functions of SHMs in all antibody genes with high efficiency. Overall, this project will provide insights into mechanisms adopted by the immune system for improving antigen-binding affinity and will provide new and general tools useful in numerous settings in antibody immunology.